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TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes

ABSTRACT: Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells are known to preserve a small pool of dormant ribosomes under nutrient-limited conditions. However, the regulation of these dormant ribosomes is poorly characterized. Here, we show that upon inhibition of yeast TORC1 by rapamycin or nitrogen starvation, the ribosome preservation factor Stm1 mediates formation of non-translating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon nutrient replenishment, TORC1 directly phosphorylates and inhibits Stm1 to reactivate translation. Finally, we find that SERBP1, a mammalian ortholog of Stm1, is likewise required for formation of dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. These data suggest that TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner by directly targeting a ribosome preservation factor.

SUBMITTER: Dr. Sunil Shetty

PROVIDER: S-SCDT-10_15252-EMBJ_2022112344 | biostudies-other |

REPOSITORIES: biostudies-other

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TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes.

Project description:Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits the degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells are known to preserve a small pool of dormant ribosomes under nutrient-limited conditions. However, the regulation of these dormant ribosomes is poorly characterized. Here, we show that upon inhibition of yeast TORC1 by rapamycin or nitrogen starvation, the ribosome preservation factor Stm1 mediates the formation of nontranslating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon nutrient replenishment, TORC1 directly phosphorylates and inhibits Stm1 to reactivate translation. Finally, we find that SERBP1, a mammalian ortholog of Stm1, is likewise required for the formation of dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. These data suggest that TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner by directly targeting a ribosome preservation factor.

| S-EPMC9975950 | biostudies-literature

TORC1 phosphorylates and inhibits the ribosome preservation factor Stm1 to activate dormant ribosomes

Project description:Target of rapamycin complex 1 (TORC1) promotes biogenesis and inhibits degradation of ribosomes in response to nutrient availability. To ensure a basal supply of ribosomes, cells preserve a small pool of dormant ribosomes under nutrient-limited conditions. The regulation of dormant ribosomes is poorly characterized. Here, we show that upon inhibition of TORC1 by rapamycin or nitrogen starvation, Stm1 (suppressor of target of Myb protein 1) forms non-translating, dormant 80S ribosomes. Furthermore, Stm1-bound 80S ribosomes are protected from proteasomal degradation. Upon re-feeding, TORC1 directly phosphorylates and inhibits Stm1, thereby reactivating translation. Finally, SERBP1 (SERPINE1 mRNA binding protein), a mammalian ortholog of Stm1, forms dormant 80S ribosomes upon mTORC1 inhibition in mammalian cells. Thus, TORC1 regulates ribosomal dormancy in an evolutionarily conserved manner via a ribosome preservation factor.

2023-01-19 | PXD037117 | Pride

Analysis of ribosome biogenesis factor-modules in yeast cells depleted from pre-ribosomes.

Project description:Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks.

| S-EPMC2875017 | biostudies-literature

TORC1-regulated protein kinase Npr1 phosphorylates Orm to stimulate complex sphingolipid synthesis.

Project description:The evolutionarily conserved Orm1 and Orm2 proteins mediate sphingolipid homeostasis. However, the homologous Orm proteins and the signaling pathways modulating their phosphorylation and function are incompletely characterized. Here we demonstrate that inhibition of nutrient-sensitive target of rapamycin complex 1 (TORC1) stimulates Orm phosphorylation and synthesis of complex sphingolipids in Saccharomyces cerevisiae. TORC1 inhibition activates the kinase Npr1 that directly phosphorylates and activates the Orm proteins. Npr1-phosphorylated Orm1 and Orm2 stimulate de novo synthesis of complex sphingolipids downstream of serine palmitoyltransferase. Complex sphingolipids in turn stimulate plasma membrane localization and activity of the nutrient scavenging general amino acid permease 1. Thus activation of Orm and complex sphingolipid synthesis upon TORC1 inhibition is a physiological response to starvation.

| S-EPMC3596256 | biostudies-literature

When ribosomes go bad: diseases of ribosome biogenesis.

Project description:Ribosomes are vital for cell growth and survival. Until recently, it was believed that mutations in ribosomes or ribosome biogenesis factors would be lethal, due to the essential nature of these complexes. However, in the last few decades, a number of diseases of ribosome biogenesis have been discovered. It remains a challenge in the field to elucidate the molecular mechanisms underlying them.

| S-EPMC2965583 | biostudies-literature

Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4.

Project description:Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.

| S-EPMC3245911 | biostudies-literature

The ribosome-associated chaperone Zuo1 controls translation upon TORC1 inhibition.

Project description:Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed of the HSP70 chaperone Ssb and its HSP40 co-chaperone Zuo1, is required to maintain proteostasis and cell viability under TORC1 inhibition in Saccharomyces cerevisiae. In the absence of Zuo1, translation does not decrease in response to the loss of TORC1 activity. A functional interaction between Zuo1 and Ssb is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Furthermore, we have shown that the rapid degradation of eIF4G following TORC1 inhibition is mediated by autophagy and is prevented in zuo1Δ cells, contributing to decreased survival in these conditions. We found that autophagy is defective in zuo1Δ cells, which impedes eIF4G degradation upon TORC1 inhibition. Our findings identify an essential role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling.

| S-EPMC10711665 | biostudies-literature

Protein kinase C-theta (PKC?) phosphorylates and inhibits the guanine exchange factor, GIV/Girdin.

Project description:G?-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein-coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, G?i1/2/3. Site-directed mutations in GIV's GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV's GEF function is regulated. Here we report that PKC?, a novel protein kinase C, down-regulates GIV's GEF function by phosphorylating Ser(S)1689 located within GIV's GEF motif. We demonstrate that PKC? specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV's ability to bind and activate G?i. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689?D, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein-coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKC? inhibits GIV's GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.

| S-EPMC3619294 | biostudies-literature

PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.

Project description:PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.

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Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria).

Project description:Akinetes are dormancy cells commonly found among filamentous cyanobacteria, many of which are toxic and/or nuisance, bloom-forming species. Development of akinetes from vegetative cells is a process that involves morphological and biochemical modifications. Here, we applied a single-cell approach to quantify genome and ribosome content of akinetes and vegetative cells in Aphanizomenon ovalisporum (Cyanobacteria). Vegetative cells of A. ovalisporum were naturally polyploid and contained, on average, eight genome copies per cell. However, the chromosomal content of akinetes increased up to 450 copies, with an average value of 119 genome copies per akinete, 15-fold higher than that in vegetative cells. On the basis of fluorescence in situ hybridization, with a probe targeting 16S rRNA, and detection with confocal laser scanning microscopy, we conclude that ribosomes accumulated in akinetes to a higher level than that found in vegetative cells. We further present evidence that this massive accumulation of nucleic acids in akinetes is likely supported by phosphate supplied from inorganic polyphosphate bodies that were abundantly present in vegetative cells, but notably absent from akinetes. These results are interpreted in the context of cellular investments for proliferation following a long-term dormancy, as the high nucleic acid content would provide the basis for extended survival, rapid resumption of metabolic activity and cell division upon germination.

| S-EPMC3280138 | biostudies-literature

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